The calcium sensing receptor (CaR) transduces local changes in extracellular calcium and metabolites (anrtino acids, polyamines) into intracellular signals, which include acute alterations in cell metabolism and secretion, and long term changes in cell proliferation and differentiation. CaR is critical to maintenance of systemic calcium levels, by controlling parathyroid PTH secretion and the balance of renal calcium excretion/resorption. Cell-specific variability in CaR signaling may in part be due to interaction(s) of CaR with scaffold proteins and the formation of cell-specific signaling complexes. The cytoskeletal protein filamin A is a scaffold for CaR, required for CaR-mediated activation of MARK signaling, and slowing CaR degradation. Preliminary data suggests CaR interactions with arrestins-1 and -2 modulate intracellular calcium responses and MARK signaling. To understand how protein scaffolds regulate CaR-mediated signaling, we will test the following hypotheses: (1) Does state-dependent arrestin-1 binding regulate CaR responsiveness? (2) Is CaR-mediated MARK signaling organized by filamin A and arrestin-2, and does the signaling pathway include transactivation of the EGF receptor? (3) Does filamin A act as a molecular escort to stabilize cellular CaR levels by protecting CaR from proteasome-mediated degradation? We will combine biochemical approaches with intracellular calcium and/or confocal imaging, using human CaR and mutant and deletion constructs. The proposed experiments will increase our understanding of the role of the protein scaffolds arrestin-1 and -2 and filamin A in enhancing the specificity of CaR signaling. More than 40 mutations in human CaR have been linked to disease, revolutionizing the clinical dissection of parathyroidisms. Allosteric modulators of CaR have clinical utility for treatment of primary hyper- and hypo- parathyroidism, secondary changes in parathyroid function resulting from renal disease, and osteoporosis. CaR stimulates cell proliferation as well as PTHrP secretion, suggesting role for CaR in potentiating the pathophysiological consequences of malignancies. Dysregulation of CaR expression and/or acute or long term signaling may, in part, be the result of altered interactions with scaffold proteins which serve to enhance CaR signaling specificity and to regulate CaR stability. Understand the role of scaffolds in CaR function may provide novel, specific sites for pharmacological intervention in treatment of calcium handling diseases. [unreadable] [unreadable] [unreadable]